Remedy for corneal failure

ABSTRACT

The present invention provides a new type of pharmaceutical agent that recovers corneal sensitivity after corneal surgery or improves the condition of dry eye. Application of a somatostatin receptor agonist is expected to provide an improvement effect on decreased corneal sensitivity after cataract surgery or LASIK surgery, decreased corneal sensitivity and dry eye associated with corneal neurodegeneration such as neuroparalytic keratopathy, corneal ulcer, diabetic keratopathy and the like.

TECHNICAL FIELD

The present invention relates to a corneal nerve axon extension promotercomprising a somatostatin receptor agonist, and an agent for therecovery or improvement of corneal sensitivity or the treatment of dryeye or corneal epithelial defect, based on the extension of cornealnerve axon.

BACKGROUND ART

Since corneal nerve is severed by corneal surgeries such as Laserphotorefractive keratectomy (PRK), Laser-Assisted-In-Situ Keratomileusis(LASIK), keratoplasy and the like, the corneal sensitivity is said todecrease generally for about 3 weeks to one year. As a result of thefunctional decrease of corneal sensitivity, patients after a cornealsurgery blink less number of times, problematically showing the symptomsof dry eye. In the patients with dry eye, lacrimal hypofunction givesrise to corneal hyposensitivity, which, upon combination with furtherlacrimal hypofunction, problematically aggravates the condition ofcorneal surface. At present, however, recovery of corneal sensitivityafter a corneal surgery is left to spontaneous recovery, and in thetreatment of dry eye, no active treatment is provided to recover cornealsensitivity.

Somatostatin is a peptide isolated from hypothalamus in 1973 as asomatotropin release inhibiting factor (SRIF), and five subtypes ofsomatostatin receptors have been found to the present day, which arerespectively named SSTR1, SSTR2, SSTR3, SSTR4 and SSTR5. Somatostatin iswidely distributed as a somatotropin release inhibitory factor in thenerve tissues, and the presence of somatostatin receptor in the eyetissue has been confirmed for iris, ciliary body and retina (Mori, M. etal., Neuroscience Letters, 1997, vol. 223, No. 3, pp. 185-188).

In addition, somatostatin has a great diversity of functions in theendocrine system, exocrine system, nerve system and the like in livingorganisms, and has been reported to be involved in, for example,neurotransmission, nerve cell growth regulation and the like, and has apromoting action on the nerve axon extension in PC12 cells (Ferriero, M.D. et., Developmental Brain Research, vol. 80, p. 13-18 (1994)).

As the eye diseases involving somatostatin, glaucoma, inflammation ofcorneal stroma, iritis, retinitis, cataract, conjunctivitis and the likeare known (JP-A-2002-515912, corresponding to WO98/58646, EP1019050).

In addition, an attempt has been made to develop somatostatin itself ora derivative thereof as a pharmaceutical product and, for example,octreotide known as a somatostatin receptor agonist is commerciallyavailable as a therapeutic drug for gastrointestinal tract hormoneproducing tumor and acromegaly•pituitary gigantism. In addition, forexample, the following are known: lanreotide (JP-A-2-289599,corresponding to EP389180), AN-238 (JP-A-2000-502055, corresponding toWO97/19954, U.S. Pat. No. 5,843,903), PTR-3173 (JP-A-2002-518339,corresponding to U.S. Pat. No. 6,051,554, U.S. Pat. No. 6,355,613), anamine derivative having affinity for SSTR2 or SSTR3 (JP-A-2000-226373,corresponding to U.S. Pat. No. 6,329,389), an aromatic amine derivativehaving a somatostatin receptor function regulating action and useful forthe prophylaxis or treatment of diabetes, obesity, diabeticcomplications and the like (JP-A-2000-191615, corresponding toEP1123918), a fused ring compound having a somatostatin receptor agonistaction and useful for the prophylaxis or treatment of diabetes and thelike (JP-A-11-209356, corresponding to U.S. Pat. No. 6,352,982), forexample, peptide having a somatostatin-like activity, which isrepresented by formula IX¹—X²-Asn-Phe-Phe-Trp-Lys-Thr-Phe-X³-Ser-X⁴wherein X¹ is Asp-Arg-Met-Pro-Cys, Arg-Met-Pro-Cys, Met-Pro-Cys, Pro-Cysor Cys, X² is Arg or Lys, X³ is Ser or Thr, and X⁴ is Cys-Lys or Cys(JP-A-10-174587),for example, a somatostatin agonist represented by formula II

and the like (JP-A-2001-518895, corresponding to WO98/45285, EP977751),for example, a somatostatin agonist represented by formula III

and the like (JP-A-2001-519811, corresponding to WO98/44921, U.S. Pat.No. 6,063,796),for example, a somatostatin agonist represented by formula IV

and the like (JP-A-2001-519812, corresponding to WO98/44922, U.S. Pat.No. 6,063,796),a somatostatin agonist represented by formula V

wherein R¹¹ is a group selected from a halogen atom, a cyano group, acarboxy group, a (C₁-C₆)alkyl group and a (C₁-C₆)alkoxy group; X is a—CH₂— group, a —SO₂— group, a —CO— group or a direct bond; and Y is a CHgroup or a nitrogen atom, and the like (JP-A-2001-114761, correspondingto EP1086947A1), for example, a somatostatin agonist represented byformula VI

and the like (JP-A-2002-3498, corresponding to US2001/047030), forexample, a somatostatin agonist acting on SSTR2, such as5-guanidino-2((2-(toluene-4-sulfonyl)-1,2,3,4-tetrahydro-isoquinoline-3-carbonyl)-amino)-pentanoicacid methyl ester and the like (US2002/91125A1),for example, a somatostatin agonist represented by formula VII

and the like (US2002/91090A1),a somatostatin analog represented by formula VIII

(WO2002/10192),an imidazolyl derivative inducing an agonist effect from one or moresubtype receptors of somatostatin (WO99/64401), a hydantoin derivativehaving affinity for somatostatin receptor (WO2001/85718), a4-aminopiperidine derivative useful as a somatostatin receptor ligand(WO2001/44191),for example, a somatostatin agonist represented by formula IX

and the like (U.S. Pat. No. 6,387,932)for example, an SSTR1 agonist represented byPhe-cyclo(Cys-D-Trp-Lys-Cys)-Thr-NH₂ (WO2000/75186),a compound represented by formula X

and the like, having a selective SSTR4 binding action and expected tohave a glaucoma treatment action (U.S. Pat. No. 6,127,343), asomatostatin analog represented by formula XI

wherein R₁ is C₁-C₄ alkyl, adamantyl and the like (WO2000/10589), forexample, a somatostatin agonist represented by formula XII

and the like (WO99/22735, corresponding to U.S. Pat. No. 6,117,880), forexample, a somatostatin agonist represented by formula XIII

and the like (JP-A-2001-502712, corresponding to U.S. Pat. No.6,020,349, U.S. Pat. No. 6,083,960),for example, a somatostatin agonist represented by formula XIV

and the like (JP-A-2001-525793 (corresponding to WO97/43278, EP912551),for example, a cyclic somatostatin analog represented bycyclo[Tyr-D-Trp-Lys-Val-Phe(4-(3-methoxyphenyl)imidazole)-Gly] and thelike (JP-A-2002-518409 (corresponding to WO99/65942, EP1086131), anergoline derivative, which is an SSTR1 selective agonist useful for thetreatment of anxiety, depression and the like (JP-A-2001-527580(corresponding to WO98/54183, U.S. Pat. No. 6,221,870), a biphenylcompound as a somatostatin receptor function regulator (JP-A-2002-80439,corresponding to WO2002/000606), a β-carboline derivative that bindswith a somatostatin receptor to block Na channel (JP-A-2002-517500,corresponding to WO99/64420, EP1086101), vapreotide (Sharon Gazal etal., Journal of Medicinal Chemistry, vol. 45, p. 1665-1671 (2002)), anamine derivative which is a compound having a somatostatin receptorbinding-inhibitory action and having a specific chemical structurecharacterized by the substitution of the 2-position of a group:

with a nitrogen atom (JP-A-2002-348287), pyridothienodiazepin havingaffinity and selectivity for a somatostatin receptor (JP-A-2002-541260,corresponding to WO2000/61587) and the like.

In contrast, there is no report on the presence of a somatostatinreceptor in cornea. With regard to the corneal nerve, moreover, a reporthas documented that most of the nerves derived from the first branch(ophthalmic branch) separating from the trigeminal ganglia aredistributed in the cornea, and deeply involved in the recovery ofsensitivity after corneal surgery, regeneration of corneal epitheliumand the like (Ke-Ping Xu et al. Cornea, vol. 15, pp. 235-239 (1996)).

However, no report has been found that teaches the presence ofsomatostatin receptor in the trigeminal nerve (corneal nerve) orpromotion of nerve axon extension of trigeminal nerve (corneal nerve) bysomatostatin.

DISCLOSURE OF THE INVENTION

The present invention provides a pharmaceutical agent that showsfunctional recovery of corneal sensitivity in patients having functionaldecrease of corneal sensitivity, from patients after corneal surgerysuch as Laser photorefractive keratectomy (PRK), Laser-In-SituKeratomileusis (LASIK), keratoplasy and the like, patients with dry eyeand the like, and that treats disorders of corneal epithelium associatedwith these functional decrease of corneal sensitivity.

The present inventors have studied for the purpose of providing a newtype of pharmaceutical agent that recovers corneal sensitivity aftercorneal surgery or improves the condition of dry eye and first foundthat somatostatin has an axon extension promoting effect for thetrigeminal nerve (hereinafter sometimes to be referred to as cornealnerve), and that a somatostatin receptor is present in the trigeminalnerve. They have further studied based on these findings, and completedthe present invention that utilizes a somatostatin receptor agonist as adrug for the recovery of corneal sensitivity and the like.

Accordingly, the present invention relates to

(1) a corneal nerve axon extension promoter comprising a somatostatinreceptor agonist,

(2) an agent for the recovery of corneal sensitivity, which comprises asomatostatin receptor agonist,

(3) a therapeutic agent for dry eye, which comprises a somatostatinreceptor agonist,

(4) a therapeutic agent for corneal epithelial defect, which comprises asomatostatin receptor agonist,

(5) a pharmaceutical composition for promoting extension of cornealnerve axon, which comprises a somatostatin receptor agonist,

(6) a pharmaceutical composition for the recovery of cornealsensitivity, which comprises a somatostatin receptor agonist,

(7) a pharmaceutical composition for the treatment of dry eye, whichcomprises a somatostatin receptor agonist,

(8) a pharmaceutical composition for the treatment of corneal epithelialdefect, which comprises a somatostatin receptor agonist,

(9) use of a somatostatin receptor agonist for the production of apharmaceutical composition for promoting extension of corneal nerveaxon,

(10) use of a somatostatin receptor agonist for the production of apharmaceutical composition for the recovery of corneal sensitivity,

(11) use of a somatostatin receptor agonist for the production of apharmaceutical composition for the treatment of dry eye,

(12) use of a somatostatin receptor agonist for the production of apharmaceutical composition for the treatment of corneal epithelialdefect,

(13) a method of promoting extension of corneal nerve axon, whichcomprises administering an effective amount of a somatostatin receptoragonist to a case in need of the promotion of extension of the cornealnerve axon,

(14) a method of recovering corneal sensitivity, which comprisesadministering an effective amount of a somatostatin receptor agonist toa case in need of the recovery of corneal sensitivity,

(15) a method of treating dry eye, which comprises administering aneffective amount of a somatostatin receptor agonist to a case affectedwith dry eye, and

(16) a method of treating corneal epithelium defect, which comprisesadministering an effective amount of a somatostatin receptor agonist toa case having corneal epithelial defect.

As used herein, by the somatostatin receptor agonist is meantsomatostatin per se, as well as those that act on somatostatin receptorand show an action similar to that of somatostatin, and encompassesthose referred to as somatostatin agonist, somatostatin analogous form,somatostatin analog and the like.

As the somatostatin receptor agonist, somatostatin per se, as well asany compound can be advantageously used as long as it acts on asomatostatin receptor and shows an action similar to that ofsomatostatin. As such compound, for example, octreotide known as asomatostatin receptor agonist, lanreotide described in JP-A-2-289599(corresponding to EP389180), octapeptides such as vapreotide and thelike, somatostatin-like cyclic peptides described in JP-A-2000-502055(corresponding to WO97/19954, U.S. Pat. No. 5,843,903), such as AN-238and the like, main chain cyclized somatostatin analog described inJP-A-2002-518339 (corresponding to U.S. Pat. No. 6,051,554, U.S. Pat.No. 6,355,613) such as PTR-3173 and the like, amine derivativesdisclosed in JP-A-226373 (corresponding to U.S. Pat. No. 6,329,389) andJP-A-2002-348287, aromatic amine derivatives described inJP-A-2000-191615 (corresponding to EP1123918), fused ring compoundsdescribed in JP-A-11-209356 (corresponding to U.S. Pat. No. 6,352,982),peptides described in JP-A-10-174587, somatostatin agonists described inJP-A-2001-518895 (corresponding to WO98/45285, EP977751),JP-A-2001-519811 (corresponding to WO98/44921, U.S. Pat. No. 6,063,796)and JP-A-2001-519812 (corresponding to WO98/44922, U.S. Pat. No.6,063,796), somatostatin agonists described in JP-A-2001-114761(corresponding to EP1086947A1), JP-A-2002-3498 (corresponding toUS2001/047030), US2002/91125A1, US2002/91090A1, U.S. Pat. No. 6,387,932,WO99/22735 (corresponding to U.S. Pat. No. 6,117,880) andJP-A-2001-502712 (corresponding to U.S. Pat. No. 6,020,349, U.S. Pat.No. 6,083,960), somatostatin analogs described in WO2002/10192 andWO2000/10589, imidazolyl derivatives described in WO99/64401, hydantoinderivatives described in WO2001/85718, 4-aminopiperidine derivativesdescribed in WO2001/44191, SSTR1 agonists described in WO2000/75186,compounds described in U.S. Pat. No. 6,127,343, which have a selectiveSSTR4 binding action and a glaucoma treatment action, somatostatinagonists as described in JP-A-2001-525793 (corresponding to WO97/43278,EP912551), cyclic somatostatin analogs described in JP-A-2002-518409(corresponding to WO99/65942, EP1086131), ergoline derivatives describedin JP-A-2001-527580 (corresponding to WO98/54183, U.S. Pat. No.6,221,870), biphenyl compounds described in JP-A-2002-80439(corresponding to WO2002/000606), β-carbolyl derivatives described inJP-A-2002-517500 (corresponding to WO99/64420, EP1086101),pyridothienodiazepin described in JP-A-2002-541260 (corresponding toWO2000/61587) and the like can be mentioned.

A pharmaceutical agent containing the somatostatin receptor agonist ofthe present invention is useful for the recovery from functionaldecrease of corneal sensitivity of cornea of mammals (e.g., human, rat,mouse, rabbit, bovine, pig, dog, cat and the like), wherein the cornealnerve is damaged or cut or the corneal epithelium is defective. Forexample, it is useful as a therapeutic drug for the recovery ofdecreased corneal sensitivity after surgery such as PRK, LASIK,keratoplasty and the like, or as a therapeutic drug for dry eye patientswith decreased corneal sensitivity, and further as a therapeutic drugfor a corneal epithelial disorder associated with functional degradationof corneal sensitivity. As the somatostatin receptor agonist, an agonistthat specifically acts on SSTR2 and/or SSTR4, which are somatostatinreceptor subtypes, is more preferable.

The pharmaceutical agent of the present invention is systemically ortopically administered. Systemically, it is orally administered, andparenterally, it is administered as intravenous injection, subcutaneousinjection, intramuscular injection and the like. Topically, it isadministered to the eye.

As the dosage form of the pharmaceutical agent of the present invention,solid agents such as powder, granule, tablet, capsule, suppository andthe like; liquids such as syrup, injection, eye drop and the like; andthe like can be mentioned. For the production of granules and tablets,any dosage form can be produced by using, for example, excipients(lactose, sucrose, glucose, starch, microcrystalline cellulose and thelike), lubricants (magnesium stearate, talc, stearic acid, calciumstearate and the like), disintegrants (starch, carmellose sodium,calcium carbonate and the like), binders (starch paste solution,hydroxypropylcellulose solution, carmellose solution, gum arabicsolution, gelatin solution, sodium alginate solution and the like) andthe like. For granules and tablets, a coating film may be formed usingsuitable coating agents (gelatin, sucrose, gum arabic, carnauba wax andthe like), enteric coatings (e.g., cellulose acetate phthalate,metacrylic acid copolymer, hydroxypropylcellulose phthalate,carboxymethylethylcellulose and the like) and the like.

For the production of capsule, a mixture of suitable excipients such asmagnesium stearate, calcium stearate, talc, light silicic anhydride andthe like for improving flowability and glidability, microcrystallinecellulose, lactose and the like for flowability under pressurization, aswell as the above-mentioned disintegrant and the like added asappropriate is uniformly admixed or granulated or granulated, coatedwith a suitable coating agent to form a film and packed in a capsule, orencapsulation-molded with a capsule base having increased plasticity,which contains a suitable capsule base (gelatin and the like), glycerinor sorbitol and the like. These capsules may contain coloring agents,preservatives [sulfur dioxide, parabens (methyl paraoxybenzoate, ethylparaoxybenzoate or propyl paraoxybenzoate)] and the like as necessary.The capsule may be a conventional one, an enteric coated capsule, agastric coated capsule or a release control capsule. When an entericcapsule is produced, a compound coated with an enteric coated agent orthe above-mentioned suitable excipients are added to a compound andpacked in a conventional capsule or a capsule itself may be coated withan enteric coating agent, or an enteric polymer may be used as a basefor molding.

For the production of a suppository, a base for suppository (e.g., cacaobutter, macrogol and the like) can be appropriately selected and used.

For the production of syrup, for example, stabilizers (sodium edetateand the like), suspending agents (gum arabic, carmellose and the like),corrigents (simple syrup, glucose and the like), aromatic and the likecan be appropriately selected and used.

For the production of the pharmaceutical agent of the present inventionas an injection or eye drop, it can be produced by dissolving ordispersing the agonist in a solution appropriately containingpharmaceutically acceptable additives such as isotonicity agents (sodiumchloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid,borax, glucose, propylene glycol and the like), buffers (phosphatebuffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer,Tris buffer, glutamate buffer, ε-aminocaproate buffer and the like),preservatives (p-oxybenzoic acid esters, chlorobutanol, benzyl alcohol,benzalkonium chloride, sodium dehydroacetate, sodium edetate, boricacid, borax and the like), thickeners (hydroxyethyl cellulose,hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol and thelike), stabilizers (sodium bisulfite, sodium thiosulfate, sodiumedetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene and thelike), pH adjusting agents (hydrochloric acid, sodium hydroxide,phosphoric acid, acetic acid and the like), and the like.

While the amount of the additives to be used for the above-mentionedsyrup, injection and eye drop varies depending on the kind of theadditives to be used, use and the like, they may be added at aconcentration capable of achieving the purpose of the additive, and anisotonicity agent is generally added in about 0.5-about 5.0 w/v % tomake the osmotic pressure about 229-about 343 mOsm. In addition, abuffer is added in about 0.01-about 2.0 w/v %, a thickener is added inabout 0.01-about 1.0 w/v %, and a stabilizer is added in about0.001-about 1.0 w/v %. A pH adjusting agent is appropriately added togenerally achieve a pH of about 3-about 9, preferably about 4-about 8.

For particular use as an eye drop, the lower limit of the concentrationof the somatostatin receptor agonist is adjusted to generally about0.0005 w/v %, about 0.001 w/v % or about 0.005 w/v % and the upper limitis adjusted to about 1.0 w/v %, about 0.5 w/v %, about 0.1 w/v %, about0.05 w/v % or about 0.01 w/v %.

While the dose of the somatostatin receptor agonist of the presentinvention varies depending on the target disease, symptom, subject ofadministration, administration method and the like, when, for example,it is topically administered to the eye of an adult after PRK surgery asan agent for the recovery of corneal sensitivity, for example, a liquideye drop containing about 0.01 w/v % of somatostatin is preferablyinstilled into the eye several times a day at about 20 to about 50 μLper dose.

When base, amino acid and the like are expressed with abbreviations inthe present description and Figures, they are based on the abbreviationsaccording to the IUPAC-IUB Commission on Biochemical Nomenclature orabbreviations conventionally used in the art. Examples thereof are shownin the following. When amino acid has an optical isomer, it is anL-isomer unless otherwise specified.

-   DNA: deoxyribonucleic acid-   cDNA: complementary deoxyribonucleic acid-   DNase: deoxyribonuclease-   SSTR: somatostatin receptor-   GAPDH: glyceraldehyde-3-phosphate-dehydrogenase-   NGF: nerve growth factor-   A: adenine-   T: thymine-   G: guanine-   C: cytosine-   RNA: ribonucleic acid-   mRNA: messenger RNA-   Gly: glycine-   Ala: alanine-   Val: valine-   Ser: serine-   Thr: threonine-   Cys: cysteine-   Met: methionine-   Asp: aspartic acid-   Lys: lysine-   Arg: arginine-   Phe: phenylalanine-   Tyr: tyrosine-   Trp: tryptophan-   Pro: proline-   Asn: asparagine-   Me: methyl group

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the expression of somatostatin receptor subtypes (SSTR2 andSSTR4) in the trigeminal nerve and retina of rabbits.

FIG. 2 shows phase contrast microscopic images of cultured rabbittrigeminal nerve cells and axon extension from the cells, wherein Ashows the cells of a non-addition group, B shows the cells of a 1 μMsomatostatin addition group, C shows the cells of a 10 μM somatostatinaddition group, D shows the cells of an NGF addition group, and E showsthe cells of a somatostatin+NGF addition group.

FIG. 3 is a graph showing the shift of the corneal sensitivity aftercutting the corneal nerve of rabbits, wherein * means a significantdifference from the vehicle administration group (n=6−12, mean±standarderror, p<0.05).

FIG. 4 shows fluorescence microscopic images showing cultured rabbittrigeminal nerve cells and axon extension from the cells, wherein Ashows the cells of a control group and B shows the cells of a 10 μMoctoreotide addition group.

FIG. 5 is a graph showing the rate (%) of neuritogenetic cells to thetotal cell number in the same test as FIG. 4, herein * shows asignificant difference (n=3, mean±standard error, p<0.05) from thecontrol.

FIG. 6 shows fluorescent microscopic images of cultured rabbittrigeminal nerve cells and axon extension from the cells, wherein Ashows the cells of a non-addition group, B shows the cells of a 1 μMcompound 1 addition group and C shows the cells of a 0.1 μM compound 2addition group.

FIG. 7 is a graph showing the absorbance indicative of the amount ofneurofilament of cultured cells (n=3, mean±standard error), in the sametest as FIG. 6.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is explained in more detail by referring to thefollowing Experimental Examples and Examples, which are not to beconstrued as limitative.

EXPERIMENTAL EXAMPLE 1

Expression of Somatostatin Receptor in the Trigeminal Nerve of Rabbit

1) Animals Used

Japanese White Rabbit (body weight 2.0 kg) purchased from FukusakiRabbit Warren was used.

2) Test Method

A mixture of Celactal (xylazine):Ketalar (ketamine hydrochloride)=0.5:1was intramuscularly injected (0.9 mL/kg) to Japanese White Rabbit forsystemic anesthesia. After cardiac perfusion with saline, retina andtrigeminal ganglia were respectively obtained. By AGPC method using aTRIzol Reagent (manufactured by GIBCO BRL), RNA was extracted from eachtissue, genome DNA was removed by DNase treatment, and a reversetranscription reaction was performed using SuperScripit II (manufacturedby GIBCO BRL). cDNA was amplified using Platinum Taq DNA polymerase(GIBCO BRL) under the reaction conditions described in Table 1. As theprimer, a rabbit somatostatin receptor SSTR2 gene specific primer (SEQID NO:1 mentioned later) and an SSTR4 gene specific primer (SEQ ID NO:2mentioned later), shown in Table 1, were used. The retina and GAPDH wereused as the positive control of the expression. TABLE 1 PCR reactionanimal gene primer (5′-3′) conditions rabbit SSTR2 TGG CCG TCT TCA TTT1.5 mM MgCl₂, pH TCT GCT CGC CGC TCA 8.4 CTT TGA CCA AG 95° C. (30 sec),(SEQ ID NO: 1) 58° C. (1 min), 72° C. (1 min) 35 cycles SSTR4 GTG GGCAAG ATG CGC 1.5 mM MgCl₂, pH GCT GTG AAT GGG GTT 10 GGC GCA GCT GTT 95°C. (30 sec), (SEQ ID NO: 2) 58° C. (1 min), 72° C. (1 min) 35 cycles3) Test Results

The results are shown in FIG. 1. The somatostatin receptor subtypesSSTR2 and SSTR4 in rabbit retina (R) and trigeminal nerve (T) wereanalyzed by RT-PCR. As a result, expression of both SSTR2 and SSTR4 wasfound in the both tissues.

EXPERIMENTAL EXAMPLE 2

Nerve Axon Extension Promoting Action in Cultured Rabbit TrigeminalNerve Cells (In Vitro Experiment)

1) Animals Used

Japanese White Rabbits (2-3 days old) purchased from Fukusaki RabbitWarren were used.

2) Test Substance

As a test substance, somatostatin (manufactured by CALBIOCHEM, LotB33795) and NGF (NGF-7S, manufactured by Sigma) were used. The testsubstance was dissolved in phosphate buffer (PBS) to 100 μMsomatostatin, 20 μg/mL NGF-7S. The prepared reagents were preserved at−80° C. and dissolved before use.

3) Test Method

The trigeminal nerve cell was isolated according to the report of Chanet al. (Kuan Y. Chan and Richard H. Haschke. Exp. Eye Res. 41: 687-699,1985). To be specific, under ether anesthesia, after cardiac perfusionof rabbit with saline, the trigeminal ganglia was removed, trigeminalganglia was dispersed using a nerve dispersion solution (SUMITOMOBAKELITE Co., Ltd.), and the cells were plated in a 24-well plate(SUMITOMO BAKELITE Co., Ltd.) coated with polylysine/laminin. The numberof cells was about 3000 cells per well and the culture conditions were5% CO₂, 95% air at 37° C. The cells were innoculated in Dulbecco'sModified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) culture mediumsupplemented with 5% fetal calf serum (FCS), cultured for 24 hr and theculture medium was exchanged to FCS-free DMEM/F-12 culture medium.

Thereafter, the cells were divided into the following 5 groups:

I non-addition group

II a group with addition of somatostatin to final concentration of 1 μM

III a group with addition of somatostatin to final concentration of 10μM

IV a group with addition of NGF to final concentration of 1 μg/mL and

V a group with simultaneous addition of somatostatin and NGF to finalconcentrations of 1 μM and 1 μg/mL, respectively, and cultured for 48hr.

The effect on the formation of neurite and the axon extension fromtrigeminal nerve cell by somatostatin and NGF were morphologicallyevaluated by the observation using a phase contrast microscope.

4) Test Results

FIG. 2 shows phase contrast microscopic images of cultured rabbittrigeminal nerve cells, wherein (A) shows the cells of group I, (B)shows the cells of group II, (C) shows the cells of group III, (D) showsthe cells of group IV, and (E) shows the cells of group V, respectively.

The formation of neurite was slightly observed in the cells of thenon-addition group (A). As compared to (A), the cells (B) of 1 μMsomatostatin addition group showed apparent promotion of axon extension,and in the cells (C) of the 10 μM somatostatin addition group, too, manycells showing long axon extension were observed. In the cells (D) of theNGF addition group and the cells (E) of NGF, somatostatin simultaneousaddition group, too, a clear nerve cell axon extension-promoting actionwas observed as compared to non-addition group (A).

EXPERIMENTAL EXAMPLE 3

Functional Changes in Corneal Sensitivity After Rabbit Corneal NerveCutting (In Vivo Test)

1) Animals Used

Male Japanese White Rabbits (body weight 1.5 kg-2.0 kg) purchased fromFukusaki Rabbit Warren were used.

2) Test Substance

As the test substance, somatostatin (manufactured by CALBIOCHEM, LotB33795) and NGF (NGF-7S, manufactured by Sigma) were used. The testsubstances were dissolved in the vehicle shown below and used for thetest.

Formulation of Vehicle sodium chloride 0.9 g sodium dihydrogen phosphatedihydrate 0.1 g sodium hydroxide suitable amount (pH 7.0) distilledwater for injection suitable amount total amount 100 mL3) Test Method

A mixture of Celactal (xylazine):Ketalar (ketamine hydrochloride)=0.5:1was intramuscularly injected (0.9 mL/kg) to the rabbits for systemicanesthesia. Cornea was labeled with trephine having a diameter of 6 mm,and incised at the upper side thereof for 180 degrees in a circle whilesuturing with a 8.0 nylon suture thread. A vehicle (vehicleadministration group, n=10), a somatostatin solution (100 μm,somatostatin administration group, n=12) or an NGF solution (20 μg/mL,NGF administration group, n=6) was instilled into the eye by 50 μL 4times/day for 4 consecutive weeks starting from immediately afteroperation. For a week after the operation, a tarivid ophthamic solution(Santen) was simultaneously instilled with the instillation of the testsubstance 4 times a day. The animals were bred in a breeding chamber setat room temperature 23±3° C., humidity 55±10%, 12 hr illumination (8:00light on, 20:00 light off) and housed at one animal per cage. Thecorneal sensitivity was measured using a Cochet-Bonnet cornealsensitivity meter (manufactured by LUNEAU) at day 3 and 1, 2, 3, 4 and 6weeks of the operation. The corneal sensitivity (%) was calculated basedon the sensitivity of each individual before operation as 100%.

4) Test Results

FIG. 3 shows the shift of corneal sensitivity with the lapse of timeafter cutting of the corneal nerve. The corneal sensitivity rapidlydecreased in all groups from the third day to one week after thekeratotomy, but mild recovery of the corneal sensitivity was found after2 weeks. Three or four weeks later, the somatostatin administrationgroup showed a significant corneal sensitivity recovery effect ascompared to the vehicle administration group (p<0.05, Dunnett's test).

EXPERIMENTAL EXAMPLE 4

Effect of Octreotide on Axon Extension of Cultured Rabbit TrigeminalNerve Cells (In Vitro Test)

1) Animals Used

Japanese White Rabbits (2-3 days old) purchased from Fukusaki RabbitWarren were used.

2) Test Substance

Octreotide (SMS 201-995, American Peptide Company, Inc.) was used.

3) Test Method

The cell culture: Trigeminal nerve cells were isolated by reference tothe report of Chan et al. (Kuan Y. Chan and Richard H. Haschke. Exp. EyeRes. 41: 687-699, 1985). To be specific, under ether anesthesia, aftercardiac perfusion of rabbit with saline, the trigeminal ganglia wasremoved, and dispersed in nerve dispersion solution (SUMITOMO BAKELITECo., Ltd.). The cells were counted and innoculated in an 8 well cultureslide (BECTON DICKINSON) coated with polylysine. The cell number wasabout 3×10³ cells per well, and the culture conditions were 5% CO₂, 95%air, 37° C. For cell culture, Neurobasal medium (GIBGO) added with B27supplement (GIBGO; 0.02 mL/mL culture solution) was used, and Octreotide(10 μM final concentration) was added to the medium immediately aftercell inoculation and the cells were cultured for 24 hr. Immunostaining:At 24 hr from culture, the cells were fixed with 4% paraformaldehyde atroom temperature for 2 hr, and nerve cell, axon and dendrite werefluorescence stained with anti-neurofilament 200 antibody (NF,Anti-Neurofilament 200, Sigma) specifically recognizing a neurofilamentconstituting the nerve cell and neurite. The images of the stained cellswere imported into a computer from the fluorescence microscope, and theeffect of Octreotide on the neurite formation and axon extension wasevaluated using an image analysis soft (MacSCOP, MITANI CO.). To bespecific, the axon extension length and the diameter of the cell weremeasured using an image analysis soft, the cells including an axonhaving a length of not less than twice the diameter of the cell weretaken as neuritogenic cells and the percentage (%) thereof to the totalcell number was calculated.

4) Test Results

FIG. 4 shows an extension effect of Octreotide on neurite and axon incultured rabbit trigeminal nerve cells, wherein (A) shows control rabbittrigeminal nerve cells cultured in an Octreotide-free medium for 24 hr,and (B) shows cells cultured for 24 hr in a medium containing Octreotideat a final concentration of 10 μM. It was confirmed that theneuritogenic cells increased in the Octreotide addition group ascompared to the control cell group.

FIG. 5 shows a proportion (%) of neuritogenic cell to the total cellnumber. The proportion of the neuritogenic cells was about 21% of thetotal cells in the control group, about 43% of the total cells in theOctreotide addition group, and a significant increase in theneuritogenic cells due to the addition of Octreotide was observed (n=3,mean±standard error, t-test, p<0.05%).

From the foregoing, it was found that Octreotide, which is asomatostatin analog, promotes the axon extension of trigeminal nervecell.

EXPERIMENTAL EXAMPLE 5

Effect of Somatostatin Receptor Agonist on Axon Extension of CulturedRabbit Trigeminal Nerve Cell (In Vitro Test)

1) Animals Used

Japanese White Rabbits (2-3 days old) purchased from KITAYAMA LABES Co.,Ltd. were used.

2) Test Substance

As a somatostatin receptor agonist, which is a test substance, t-butyl6-amino-2-(3-(1H-indol-3-yl)-2-((4-(2-oxo-2,3-dihydrobenzoimidazol-1-yl)piperidine-1-carbonyl)amino)propionylamino)hexanoate,which is an SSTR2 specific agonist (hereinafter to be referred to ascompound 1), and1-(3-(N-(5-bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino)propyl)-3-(3-(1H-imidazol-4-yl)propyl)thiourea(hereinafter to be referred to as compound 2), which is an SSTR4specific agonist, were used. The compound 1 was a compound of Example 4and synthesized according to the method of Example 2 of JP-A-2001-519812(WO98/44922). The compound 2 was synthesized according Example 15 ofJP-A-2001-525793 (WO97/43278).

3) Test Method

Cell Culture:

Rabbit trigeminal nerve cells were isolated by reference to the reportof Chan et al. (Kuan Y. Chan and Richard H. Haschke. Exp. Eye Res. 41:687-699, 1985). To be specific, under ether anesthesia, cardiacperfusion with saline was performed and trigeminal ganglia was removed.Using nerve dispersion solution (SUMITOMO BAKELITE Co., Ltd.),trigeminal ganglia was dispersed to prepare trigeminal nerve cells. Forcell culture, Neurobasal medium (GIBCO) added with B27 Supplement(GIBCO; final concentration 2% v/v) and L-Glutamine (GIBCO; finalconcentration 1 mM) was used under the culture conditions of 5% CO₂, 95%air, 100% humidity, 37° C., 48 hr. The cells were innoculated on an 8well culture slide (BECTON DICKINSON) coated with polylysine at about3×10³ cells/well, or on a 96 well plate coated with polylysine at 3×10³cells/well and the medium of the test substance group contained compound1 (final concentration 1 μM) or compound 2 (final concentration 0.1 μM).

Cell Staining:

After the completion of 48 hr culture, the cells innoculated on a 96well plate were fixed with 4% paraformaldehyde and the neurofilament ofthe cell was labeled with anti-neurofilament 200 antibody (Sigma)specifically recognizing a neurofilament constituting nerve cell bodyand neurite and HRP conjugated goat anti-mouse IgG antibody (Wako PureChemical Industries, Ltd.). Onto the labeled cells was added a citratebuffer containing 50 μL of 0.02% H₂O₂ (Nacalai Tesque) and 0.2%o-phenylenediamine (Sigma) to allow color development for 30 min. Then,50 μL of 4.5 M H₂SO₄ (Nacalai Tesque) was added to quench the reaction.After the completion of coloring, the absorbance at 492 nm was measuredand the measured value was taken as the amount of the stainedneurofilament and used as an index for neuritogenesis (Taniwaki T., etal. Dev. Brain Res. (1995) 88, 109-166). The cells plated on an 8 wellculture slide were fixed with 4% paraformaldehyde, a sample fixed withan anti-neurofilament 200 antibody (Sigma) was stained, and fluorescencelabeled with Alexa Fluor 568 conjugated secondary antibody (Molecularprobes). The fluorescence labeled cells were observed under afluorescence microscope and the cell images were imported into acomputer.

4) Test Results

FIG. 6 shows fluorescence microscopic images of cultured rabbittrigeminal nerve cells, wherein (A) shows the cells of the control groupcultured in a somatostatin receptor agonist-free medium, (B) shows thecells of the group with the addition of compound 1 at a finalconcentration of 1 μM, and (C) shows the cells of the group with theaddition of compound 2 at a final concentration of 0.1 μM. As comparedwith the cells of the control group, the cells of the group with theaddition of compound 1 or compound 2 were confirmed to show an increasein the neuritogenic cells. FIG. 7 is a graph showing the absorbanceindicating the neurofilament amount of the cells of each addition group,wherein the absorbance of the control group was 0.798, and theabsorbance of the compound 1 addition group and compound 2 additiongroup was 0.876 and 0.850, respectively. That is, the amount ofneurofilament increased depending on the addition of the somatostatinreceptor agonist, which is considered to reflect the increase in theneuritogenic cells.

From the foregoing, it was found that compound 1 and compound 2, whichare somatostatin receptor agonists, have an action to promote axonextension of trigeminal nerve cell.

Formulation Examples are shown in the following.

EXAMPLE 1

Tablet somatostatin 50 mg lactose 80 mg starch 17 mg magnesium stearate3 mg Microcrystalline cellulose 10 mg

Using the above components as a material for one tablet, tablets aremolded according to a conventional method. The tablets may be coated asnecessary with a conventional enteric coating (e.g.,hydroxypropylmethylcellulose phthalate and the like), or a sugar coatingor film (e.g., ethylcellulose).

EXAMPLE 2

Capsule somatostatin 75 mg mannitol 75 mg starch 17 mg calcium stearate3 mg

Using the above components as a material for one capsule, they areuniformly mixed, granulated according to a conventional method andpacked in a hard capsule. Before packing, the granules may be coated asnecessary with a conventional enteric coating (e.g.,hydroxypropylmethylcellulose phthalate), sugar coating or film (e.g.,ethylcellulose).

EXAMPLE 3

Injection Compound 1 750 mg carboxymethylcellulose sodium 500 mg waterfor injection suitable amount total amount 100 mL

The above components were aseptically admixed according to aconventional method to give an injection.

EXAMPLE 4

Eye Drop Compound 2 50 mg boric acid 700 mg borax suitable amount (pH7.0) sodium chloride 500 mg hydroxymethylcellulose 0.5 g sodium edetate0.05 mg benzalkonium chloride 0.005 mg sterilized purified watersuitable amount total amount 100 mL

Sterilized purified water (80 mL) was heated to about 80° C.,hydroxymethylcellulose was added and the mixture is stirred until theliquid temperature reaches room temperature. Compound 2, sodiumchloride, boric acid, sodium edetate and benzalkonium chloride are addedto this solution to allow dissolution. A suitable amount of borax isadded to adjust the pH to 7. Sterilized purified water is added tomeasure up to 100 mL.

EXAMPLE 5

Eye Drop acetic octoreotide 112 mg D-mannitol 4.5 g sodium dihydrogenphosphate 0.1 g sodium hydroxide suitable amount (pH 7.0) sterilizedpurified water suitable amount total amount 100 mL

Acetic octoreotide, D-mannitol and sodium dihydrogen phosphate are addedto sterilized purified water (80 mL) to allow dissolution. A suitableamount of sodium hydroxide is added to adjust the pH to 5.0. Sterilizedpurified water is added to measure up to 100 mL. The prepared eye dropis aseptically filtered with a membrane filter and filled in adisposable (unit dose) container and sealed.

INDUSTRIAL APPLICABILITY

Since the pharmaceutical agent of the present invention, which containsa somatostatin receptor agonist, has a trigeminal nerve cell axonextension promoting action and an action to functionally recoverdecreased corneal sensitivity, it is useful for improving functionaldecrease of corneal sensitivity associated with corneal nerve damage andthe like, and the symptoms of dry eye associated with functionaldecrease of corneal sensitivity. Specifically, application of asomatostatin receptor agonist is expected to provide an improvementeffect on decreased corneal sensitivity after cataract surgery or LASIKsurgery, decreased corneal sensitivity and dry eye associated withcorneal neurodegeneration such as neuroparalytic keratopathy, cornealulcer, diabetic keratopathy and the like.

While some of the embodiments of the present invention have beendescribed in detail in the above, it will, however, be evident for thoseof ordinary skill in the art that various modifications and changes maybe made to the particular embodiments shown without substantiallydeparting from the novel teaching and advantages of the presentinvention. Such modifications and changes are encompassed in the spiritand scope of the present invention as set forth in the appended claims.

This application is based on a patent application Nos. 18881/2002 and040250/2003 filed in Japan, the contents of which are herebyincorporated by reference.

1-12. (canceled)
 13. A method of promoting extension of corneal nerveaxon, which comprises administering an effective amount of asomatostatin receptor agonist to a case in need of the promotion ofextension of the corneal nerve axon.
 14. A method of recovering cornealsensitivity, which comprises administering an effective amount of asomatostatin receptor agonist to a case in need of the recovery ofcorneal sensitivity.
 15. A method of treating dry eye, which comprisesadministering an effective amount of a somatostatin receptor agonist toa case affected with dry eye.
 16. A method of treating cornealepithelium defect, which comprises administering an effective amount ofa somatostatin receptor agonist to a case having corneal epithelialdefect.